The present proposal focuses on evaluation and optimization of vaccine strategies using defined, shared melanoma peptides from the melanocytic differentiation proteins (MDPs) and cancer-testis families of proteins. In studies performed to date, we have observed immunogenicity of MDP peptides after vaccination. Challenges for optimization of peptide-based tumor vaccines include the need to optimize the magnitude of the CTL response and the need to increase the number of antigens being targeted. Successful completion of the work proposed here will contribute to optimization of peptide vaccine strategies in these areas, specifically addressing the following aims: Aim 1 will investigate the biologic effects of a combination adjuvant on dendritic cells at the site of cutaneous vaccination and at the draining lymph nodes. This combination adjuvant includes GM-CSF and incomplete Freund's adjuvant, and the value of GMCSF in that adjuvant will specifically be evaluated. Aim 2 will include a clinical trial that will determine whether a set of 12 peptide epitopes for melanoma-reactive CTL are immunogenic in humans when incorporated into a vaccine as free peptides and whether this 12 peptide vaccine is more effective at inducing T-cell responses than the 4 peptide vaccine. Aim 3 will determine whether a set of 7 defined epitopes for melanoma-reactive T-helper cells are immunogenic in humans and whether they synergize to increase the magnitude of the response to CTL epitopes. Thus, the current proposal represents a continued evolution of our laboratory effort into the realm of patient-oriented research founded in basic tumor immunology. We expect that the lessons learned from optimization of peptide-based vaccine strategies will be relevant both for continued development of these approaches and also for tumor vaccines of other types. The work proposed in this application will set the stage for randomized trials comparing optimized peptide vaccines to optimized vaccines of other types.